Neural networks in the brain, which generate fundamental motor behaviors such as walking and breathing, are hypothesized to consist of coupled rhythmic networks that can be reconfigured to produce different motor behaviors. The long-range goal is to determine how coupling and reconfiguration in rhythmic networks contribute to motor behavior in mammals. Highly versatile 'microfluidic chambers' will be constructed (of biocompatible polydimethylsiloxane [PDMS]) to study brain slices in vitro. These 'closed-top' and 'open-top' chambers will permit spatiotemporal control of slice extracellular space combined with a variety of neural recording configurations (e.g., 2-D and 3-D multichannel extracellular, intracellular, and suction electrodes). Transverse slices of neonatal rat medulla will be used because these slices spontaneously produce rhythmic respiratory-related motor output in these chambers for hours. Medullary slices are also ideal because the rhythmic motor networks that generate respiratory-related motor output (i.e., the pre-Botzinger Complex) are synaptically-coupled and located bilaterally in slices (i.e., spatially separate). In each type of chamber, multiple parallel fluid streams exhibiting laminar flow will pass over (and under) the slice. Since laminar flow prevents fluid streams from mixing, the composition of fluid bathing the slice in specific regions will be altered by controlling laminar stream composition and width. In both closed-top and open-top chambers, Aim 1 will establish how different factors affect laminar flow and diffusion in slices, and establish the microelectrode conformation that best captures network activity. Aim 2 will show that focused laminar streams can: (a) reversibly uncouple bilateral rhythmic motor networks (with sodium-free sucrose solution), and (b) reconfigure one rhythmic motor network (with hypoxic solution) while it is coupled to another rhythmic motor network. Microfluidic chambers will provide a novel approach to neural circuit analysis, which is a major goal of NINDS. Innovative studies on network coupling and reconfiguration may lead to therapeutic insights for treating spinal cord injury, stroke, cerebral palsy, and Parkinson's disease. Microfluidic chambers can also be modified to perform novel studies on synaptic and network plasticity, effects of local ischemia or hypoxia, intracellular signaling networks, and cell-based biosensing. [unreadable] [unreadable] [unreadable]